Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis

Rachel K Carver-Brown,Arthur H Reis,Lawrence J Wangh,Lisa M Rice,John W Czajka

doi:10.1155/2012/424808

Rachel K Carver-Brown, Arthur H Reis + Show 3 more

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https://doi.org/10.1155/2012/424808

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Abstract

Aims. The goal of this study was to construct a single tube molecular diagnostic multiplex assay for the detection of microbial pathogens commonly associated with septicemia, using LATE-PCR and Lights-On/Lights-Off probe technology. Methods and Results. The assay described here identified pathogens associated with sepsis by amplification and analysis of the 16S ribosomal DNA gene sequence for bacteria and specific gene sequences for fungi. A sequence from an unidentified gene in Lactococcus lactis subsp. cremoris served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were then analyzed at endpoint over a wide temperature range in a specific fluorescent color. Each bacterial target was identified by its pattern of hybridization to Lights-On/Lights-Off probes derived from molecular beacons. Complex mixtures of targets were also detected. Conclusions. All microbial targets were identified in samples containing low starting copy numbers of pathogen genomic DNA, both as individual targets and in complex mixtures. Significance and Impact of the Study. This assay uses new technology to achieve an advance in the field of molecular diagnostics: a single-tube multiplex assay for identification of pathogens commonly associated with sepsis.

Highlights

Sepsis affects millions of people, killing at least 1 in 4 [1, 2]
We have optimized linear-after-the-exponential PCR (LATE-PCR) to overcome the challenges inherent to construction of a highly informative single-tube multiplex assay for septicemia where an unlimited number of possible pathogens must be detected
In this design the 16S bacterial genomic DNA was detected with the Lights-On/Lights-Off probes in the Quasar 670 fluorescent dye channel while the Lactococcus lactis subsp. cremoris control DNA and Candida species of interest were detected in the Cal Org 560 and Cal Red 610 channels, respectively

Summary

Introduction

Sepsis affects millions of people, killing at least 1 in 4 [1, 2]. Nucleic-acid-testing- (NAT-) based methods of pathogen identification have for some time been regarded as a desirable alternative to conventional culture-based diagnostic methods for analysis of septicemia specimens [3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23]. LATE-PCR is a form of nonsymmetric PCR that generates single-stranded DNA making it possible to probe and quantify these strands at endpoint [33,34,35,36,37,38,39,40,41]. Utilizing both the expanded temperature and fluorescent space available for detection of targets at endpoint, LATE-PCR assays can readily be multiplexed [40]

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Conclusion